Structure and function of the bacterial primosome

描述(由申请人提供)：DNA复制重新启动路径将DNA复制机制重新加载到由于基因组损伤而被放弃的复制叉子上。这些途径在断裂复制叉的修复(通常是重组修复)和DNA复制之间形成了重要的生化联系。驱动这些反应的蛋白质，称为原始体或复制重新启动蛋白(RRP)，必须识别这些被遗弃的复制叉子的结构，并在这些位置重新加载DNA复制机制。这一过程受到严格的监管，以确保装载的保真度，并避免因在不正确的DNA结构上启动复制而可能产生的过度复制。这个 DNA复制重新启动的结构机制以及它与其他细胞基因组维护过程整合的细胞机制目前尚不清楚。我们的建议结合了结构、生化和遗传方法，以互补的方式定义DNA复制重新启动途径的机制。我们将使用X射线结晶学来确定组成原始体的关键蛋白质和蛋白质复合体的晶体结构(目标1)。这些研究将产生分子模型，有助于定义细菌RRP发挥作用的物理机制。此外，我们将从生物化学的角度定义RRP如何相互作用以驱动复制重新启动(目标2)。这组实验将把目标1中产生的物理模型与复制重新启动路径上的步骤联系起来，以揭示原始体是如何将复制叉子识别与RRP复杂组装联系在一起的。最后，我们将确定在细菌细胞中协调复制重新启动与DNA复制、重组和修复过程的联系(目标3)。这些连接将有助于确定如何将复制重新启动整合到细胞中的基本基因组维护网络中，以及如何管理其使用以防止不必要的复制启动。 公共卫生相关性：从人类健康的角度来看，DNA复制重新启动是一个重要的研究过程，因为更多地了解DNA复制将有助于识别靶点，选择性地杀死可能困扰我们的有害、恶性或引起疾病的细胞 身体(例如，肿瘤或病原体)。相反，这些知识也可以用来增强我们细胞的基本健康和寿命。这可能会转化为个人更长、更健康的寿命。

DESCRIPTION (provided by applicant): The DNA replication restart pathways reload the DNA replication machinery onto replication forks that have been abandoned as a consequence of genomic damage. These pathways form an essential biochemical link between repair (often recombinational repair) of broken replication forks and DNA replication. The proteins that drive these reactions, referred to as the primosome or the Replication Restart Proteins (RRPs), must recognize the structures of these abandoned replication forks and reload the DNA replication machinery at these sites. This process is heavily regulated to ensure loading fidelity and to avoid over-replication that could arise from initiating replication at improper DNA structures. The structural mechanisms underlying DNA replication restart and the cellular mechanisms by which it is integrated with other cellular genome maintenance processes are currently poorly understood. Our proposal combines structural, biochemical, and genetic approaches to define the mechanisms of DNA replication restart pathways in complementary ways. We wil use X- ray crystallography to determine the crystal structures of key proteins and protein complexes that comprise the primosome (Aim 1). These studies will produce molecular models that will help define the physical mechanisms by which bacterial RRPs function. Additionally, we will define biochemically how RRPs interact with one another to drive replication restart (Aim 2). This set of experiments will link the physical models generated in Aim 1 to steps along the replication restart pathways, to reveal how the primosome ties replication fork recognition to RRP complex assembly. Finally, we will identify linkages that coordinate replication restart with DNA replication, recombination, and repair processes in bacterial cells (Aim 3). These connections will help define how replication restart is integrated into the basal genome maintenance network in cells and how its use is regulated to prevent unwarranted replication initiation. PUBLIC HEALTH RELEVANCE: DNA replication restart is an important process to study from a human health perspective because knowing more about DNA replication will help to identify targets to kill selectively the deleterious, malignant or disease causing cells that may plague our bodies (e.g., tumors or pathogenic organisms). Conversely, this knowledge may also be used to augment the basic health and longevity of our cells. This may be translated into a longer, healthier life for the individual.